Type I collagen is the main component in the extracellular matrix (ECM) of the lungs. Several important diseases such as asthma and interstitial pulmonary fibrosis involve detrimental collagen production and ECM remodeling. In addition, there are several reports in the literature of important interactions between the epithelium and the fibroblast in modulating the ECM. We studied the ECM of a fibroblast-imbedded collagen gel designed as an in vitro model of the interstitial connective tissue of the lung. The model system was a 3-D co-culture consisting of human lung fibroblasts (CCD-18 lu), denatured type I collagen, fetal bovine serum (FBS), sodium hydroxide, fibroblasts, media, and a monolayer of human alveolar epithelial cells (A549). The gels were fixed at 0, 3, and 6 day intervals, and observed by immunohistochemical staining, transmission electron microscopy (TEM), and two-photon microscopy (TPM). Two cases were studied: 1) gels cultured in media supplemented with 10% serum, and 2) serum-free culture. In both cases, gels cultured without the monolayer served as a control. Preliminary analysis of live cultures shows the presence of fibers/bundles displaying TPM-induced autofluorescence signatures consistent with collagen and/or elastin. In medi a supplemented with 10% serum, matrix fibers were observed from Day 3 and increased in number through Day 6. Similarly, in serum-free culture the number of detectable fibers increased from Day 3 through Day 6. However, the total number of bundles per day was less than that of the gels in media with serum. We conclude that human lung fibroblasts remodel the ECM in vitro, and that factors present in serum (i.e., TGF-b1) enhance the remodeling process.